Staphylococcus aureus is an opportunistic pathogen that causes a broad spectrum of acute and chronic infections. Antibiotic resistance is a growing challenge and methicillin-resistant S. aureus (MRSA) infections are more difficult to treat, resulting in increased burden for both patients and healthcare systems. S. aureus causes the majority of skin infections in civilians and the military, but how this pathogen colonizes the skin is unknown. In recent microscopy studies on skin explants, S. aureus developed biofilm communities during skin colonization, and these biofilms were found to produce virulence factors under control of the agr quorum-sensing system. In our preliminary studies, we found that MRSA strains lacking agr show striking defects in skin explant colonization, and in rodent skin colonization models. Our central hypothesis is that MRSA quorum-sensing is essential for skin colonization and evasion of host defenses. Additionally, we believe quorum-sensing plays a critical role in the transition from skin to systemic infection. In Specific Aim 1, we will determine the role of quorum- sensing during MRSA skin colonization. We hypothesize that MRSA strains use agr-regulated factors to colonize the skin. To address this hypothesis, we will compare MRSA WT and ?agr mutant strains using in vitro models of adherence and compare them in a mouse skin colonization model. We will also identify agr- regulated factors required for colonization and determine whether known biofilm factors are important. Lastly, we will perform RNAseq to obtain a broader assessment of MRSA regulated functions on skin. In Specific Aim 2, we will determine the contribution of quorum-sensing to MRSA immune evasion on the skin. We hypothesize that MRSA evades skin immunity using agr-regulated factors. Toward this end, we will determine the role of quorum-sensing in the induction and resistance to antimicrobial peptides and assess the quorum-sensing response to skin unsaturated fatty acids. We will also determine whether MRSA urease and other agr-regulated factors contribute to pH homeostasis, and we will evaluate the host skin response by single-cell RNAseq. In Specific Aim 3, we will assess the function of quorum-sensing in dissemination from colonization. We hypothesize that the MRSA quorum-sensing is required for systemic dissemination from the skin. To further investigate this mechanism, we will determine the requirement for MRSA quorum-sensing function and agr-regulated factors in skin dissemination to distal sites. We will also perform real-time imaging of infections and determine the quorum-sensing contribution to evasion of host immunity. An improved understanding of how MRSA colonizes the skin and transitions to infection could open avenues to developing therapeutic strategies for minimizing the skin infection burden.